This is invention relates to a method of identifying candidate contrast agents (xe2x80x9ccontrast agent drug candidatesxe2x80x9d) using combinatorial libraries of compounds or molecular aggregates.
Traditionally, drug discovery has involved the manufacture and testing of individual compounds, often as a result of an initial fortuitous finding of efficacy for a naturally occurring compound and the subsequent synthesis of structurally similar compounds. Recently however an alternative approach to drug discovery has developed in which a large range of compounds of random or directed random structure are synthesised and tested for efficacy. In such xe2x80x9ccombinatorialxe2x80x9d libraries, the library members may either be separate or be present in admixture. The present invention is concerned with the latter case.
Such admixture libraries may readily be tested for efficacy in vitroxe2x80x94however if the desired drug effect is found, drug candidate identification requires deconvolution, the identification of which member or members of the library have the desired effect. Several deconvolution techniques have been developed and particular mention here may be made of biological tagging and orthogonal scanning. In biological tagging, the individual library members carry a unique biological tag that can be amplified, e.g. an oligonucleotide sequence or a virus, so enabling the xe2x80x9csuccessfulxe2x80x9d library member, once isolated from the unsuccessful members, to be identified. In orthogonal scanning, numbers of sub-libraries are screened in order to identify the xe2x80x9csuccessfulxe2x80x9d library members.
Screening of combinatorial libraries containing the library members in admixture is generally done on an in vitro basis with in vivo testing being left until potential drug candidates have been identified and then being performed using individual drug candidates, much as in conventional drug discovery.
In vitro screening however is of limited applicability since it requires a sample of the target tissue or a culture of target cells to be used and thus it does not allow one to determine whether or not the xe2x80x9csuccessfulxe2x80x9d library members associate specifically with the target and it does not allow one to determine targeting ability for unknown targets.
It is possible to screen mixed combinatorial libraries in vivo where the library members carry a biological, amplifiable tag. The xe2x80x9csuccessfulxe2x80x9d library members may then be identified by excision of tissue from the desired target site, amplification and identification. Again however this has drawbacks since the removal of tissue from the animal, and often the sacrifice of the animal, is required and thus this technique is generally suitable only for small laboratory animals such as mice, guinea pigs and rats. Accordingly, it does not generally allow one to determine targeting ability and specificity for unknown targets in larger, e.g. more human-like, animals.
These drawbacks however can be overcome by in vivo screening of a mixed combinatorial library in which the library members incorporate reporters which are detectable and interdistinguishable in vivo.
Thus viewed from one aspect the invention provides a method of contrast agent drug candidate selection which involves:
(i) obtaining a combinatorial library comprising an admixture of potential contrast agent drug candidates each incorporating a reporter moiety which is detectable in the animate human or non-human animal body (e.g. mammalian, avian or reptilian body), said library comprising a plurality of said reporter moieties which are interdistinguishably detectable in said body,
(ii) administering said library to an animate human or non-human animal body,
(iii) identifying in vivo one or more of said reporter moieties having a desired distribution and/or elimination pattern (e.g. which distributes to a particular site in said body) and thereby identifying a member of said library which has said pattern (e.g. which distributes to said site) or a sub-set of said library which contains a member of said library which has said pattern (e.g. which distributes to said site).
For the candidate contrast agents to be deemed xe2x80x9csuccessfulxe2x80x9d, ie. to progress to a further stage of development, they must possess a desired pattern of biodistribution and bioelimination following administration. This may be referred to by the acronym xe2x80x9cADMExe2x80x9d standing for administration, distribution, (metabolism) and excretion. The method of the present invention serves to identify members of the combinatorial library or subsets within the combinatorial library that have an ADME pattern which meets the selection criteria set down for contrast agent candidate identification.
The library members in the combinatorial library used according to the invention incorporate a reporter moiety which is detectable in an animate body. By this it is meant that the reporter should be such as to allow its detection without requiring removal (ie. separation) from the body of a sample of a body site of interest and so also without requiring the animal to be sacrificed. Examples of reporter moieties detectable directly in this fashion include chromophores, heavy atoms, radiolabels and magnetic particles, ie. moieties such as are commonly used for image contrast enhancement in diagnostic imaging modalities such as light imaging, X-ray imaging, magnetic resonance imaging, ultrasound imaging and nuclear imaging.
However it is central to the invention that the library should contain a plurality of interdistinguishable reporters, ie. reporters which can not only be detected in the animate body but which can be distinguished from each other in such detection. Particularly desirably, each different library member has a different reporter so that detection of a reporter identifies the library member it forms part of. In this case the number of different reporters is equal to the number of different library members. However in practice it will often be preferable to have a smaller number of different reporters than different library members so that detection of a reporter identifies a sub-set of the library members that include that particular reporter. In this case, with the sub-set identified, orthogonal scanning techniques can be used to identify the xe2x80x9csuccessfulxe2x80x9d members of the initial library. Conventional deconvolution techniques may be used in this regard. Thus for example a series of libraries may be scanned in which the reporters used to tag the library members are varied with the successful members being identified by a unique combination of reporters detected in the series of scans. Alternatively a sub-library may be prepared using the members of the initially identified library sub-set but labelling these with a plurality of different reporters. A second scan can then be used to identify either the successful library member or a smaller sub-set containing it (in which latter case the procedure is repeated to identify the successful library member).
The interdistinguishability of the reporters may be achieved in a variety of ways. Thus for example reporters may be used which are detectable by different imaging techniques (in which case screening will involve imaging the body or a site of interest therein using two or more imaging techniques) or alternatively or additionally reporters may be used which are interdistinguishable in one imaging technique. In this regard, particular mention may be made of radiolabels which emit detectable radiation of different types or energies detectable by multichannel gamma counters, of chromophores with different characteristic absorption or emission maxima, of tags with different detectable radiofrequencies (see for example Nicolaou et al. Angew. Chem. Int. Ed. Engl. 34: 2289-2291 (1995)), of tags with different magnetic moments (preferably using different monosized crystals, optionally differentiating with SQUID magnetometers, or Mxc3x6ssbauer or other spectroscopie techniques), of tags with different paramagnetic centres (e.g. differentiating using epr), or tags with differently sized or membraned gas containing microballoons. In a further embodiment of the invention a combination of reporters which may be interdistinguishable in one or more imaging techniques may be incorporated in individual members of the combinatorial libraries.
For ease of operation, it will be preferred that the reporters used be detectable in a single imaging modality, or less preferably up to 5 imaging modalities.
Direct means of contrast agent direction may involve a combination of detection techniques, e.g. HPLC-MS, MS-MS, capillary electrolysis-mass spectroscopy (CE-MS), HPLC-NMR, etc.
In order to avoid the situation where differences in targeting ability of the library members may not be determinable due to the differences in modification of targeting ability caused by different chemical or physical properties of the different reporter tags used, it may be desirable to use only chemically/sterically similar reporters in each library. Thus for example the reporters used in a library might all be phthalocyanines, with each different reporter having a characteristic absorption or emission maximum.
The reporter species mentioned above are all directly detectable. However in the present invention it is also possible to use indirectly detectable reporters, for example materials which become detectable or produce a detectable material through interaction with a further material or materials which modify the detectability of a further material. Thus for example a reporter might be a specific binding partner (e.g. an oligopeptide or oligonucleotide) for a component of a detectable material (e.g. a contrast agent comprising a magnetic particle or a gas containing vesicle) which could be administered after the library has been administered and after unsuccessful library members have been cleared from the site of interest. Since a vast number of complimentary binding partners may be produced relatively easily and since the library members do not need to contain a directly detectable moiety which might adversely affect their biodistribution, this embodiment offers a simple solution to the production of a deconvolutable library usable in vivo without requiring animal sacrifice.
The method of the present invention will generally involve the use of at least one combinatorial library having at least 3 library members, preferably at least 10, more preferably at least 50 members and still more preferably at least 100 members. In certain cases the libraries used may contain significantly larger numbers of members, e.g. up to 104 or even up to 106. The smaller the number of library members the more important it will be to use rational rather than entirely random design of the library members in order to have a reasonable expectation of finding a xe2x80x9csuccessfulxe2x80x9d member of the library. The concentration of each library member in the library likewise will depend upon the detectability of the reporters used as well as upon the means of administration and whether or not a target site of interest has already been identified. In general however, as in most combinatorial chemistry library screens, it will be desirable for the library used to contain multiple copies of the library members.
The number of different, interdistinguishable reporters present in the library will also depend upon a number of factors. Thus while ideally each library member is uniquely tagged, in certain cases the nature of the selected reporter moieties may not permit unique tagging of the members of a relatively large library. While there will be at least two different reporters, preferably there will be at least 5, more preferably at least 10 and especially preferably at least 25. Put otherwise, the ratio of library members to different reporters will ideally be 1:1 but may be as low as 10000:1 with ratios in the range 1:1 to 11000, 1:1 to 1:200, more especially 1:1 to 1:50 and even more especially 1:1 to 1:25 being acceptable.
Where library members are not uniquely tagged, deconvolution techniques may be used as mentioned above to identify the successful library member(s) or at least to identify a smaller library sub-set in which the successful library member may be found. If a small enough sub-set is identified then the final identification of the successful library member need not involve further deconvolution but might instead involve individual testing of members of that sub-set and if desired may involve removal of samples and if required animal sacrifice.
Identification of successful library members may be sufficient to allow selection of a drug candidate. However, especially where library members have been generated on a random basis, identification of successful and unsuccessful members may permit the generation of further libraries with an increased expectation of containing successful members. Thus for example where the library members are oligomers or are framework decorated monomers produced by reaction of differently substituted precursor molecules (e.g. amino acid or nucleotide monomers or the xe2x80x9cjigsaw piecexe2x80x9d reagents used to construct a framework decorated monomer), then monomers, monomer strings or jigsaw piece reagents which occur in unsuccessful but not successful library members may be excluded and further analogs of monomers, monomer strings or jigsaw piece reagents which occur in successful but not unsuccessful library members may be added in the synthesis of the subsequent library. Typically, iterative library redesign might be carried out up to 10 or even more times in order eventually to reach a drug candidate suitable for selection.
The method of the invention may thus involve screening of a plurality of libraries, e.g. 2 to 20, preferably 2 to 10, with successive libraries being designed on the basis of the results from the earlier libraries and iteration being stopped when one or more conjugates having suitable properties are found.
As mentioned above, the reporters used need not be directly detectable. It is also not essential that they be detectable (directly or indirectly) at the site within the body at which they accumulate. Thus for example the body can be used to separate successful from unsuccessful library members with the successful members (or subsets) being identified by elimination by identification of the unsuccessful members (or subsets) or on release from their accumulation site, for example by administration of a ligand for that site. In effect this means that the animal body is used as a means for chromatographic separation, e.g. affinity chromatographic separation, of the library members.
In this aspect of the invention, it is not necessary that the reporter be detectable within the animate body as the detection may be effected using a fluid sample from the body (e.g. a blood, urine, CSF, bile, etc. sample) which can be taken for analysis without requiring the animal to be damaged or sacrificed. Since a fluid sample may be taken for analysis, it is especially preferable that, where the fluid sample is one expected to contain a successful library member, the reporter used be one which is capable of amplification, e.g. an oligonucleotide or an organism (for example a phage), and that the library members be uniquely tagged.
Thus viewed from a further aspect the invention provides a method of contrast agent drug candidate selection which involves:
(i) obtaining a combinatorial library comprising an admixture of potential contrast agent drug candidates each incorporating a detectable reporter moiety, said library comprising a plurality of said resorter moieties which are interdistinguishably detectable,
(ii) administering said library to an animate human or non-human animal body,
(iii) preferably without sacrificing said animal, obtaining body fluid from said body and identifying one or more of said reporter moieties present or not present in said body fluid and thereby identifying a member of said library or a subset of said library which contains a member of said library which is present or not present in said body fluid.
In this method, step (iii) may if desired be effected repeatedly or continuously in order to determine the time dependence of the appearance in the obtained body fluid of the library members or subsets. For certain body fluids, such as blood or CSF, the obtained fluid may be continuously returned to the body after analysis, ie. analysis may be performed ex vivo.
In this method of the invention, after the library has been administered and after sufficient time has elapsed for a separation of successful and unsuccessful library members to have occurred in the body, a material which binds to a site of interest (e.g. a cell surface receptor) may be administered so as to displace successful library members which have bound to that site whereafter such displaced successful library members may be detected in the body fluid in question, e.g. blood or urine.
The methods of the invention are applicable to library screening in vivo using an animate animal subject. However they may also be carried out using isolated but living organs, for example the heart. This aspect of the invention is particularly applicable where the reporters used are difficult to detect with the desired spatial discrimination in the live animal or where the intention is to identify materials whose ability to accumulate at particular sites of interest is or is not affected by abnormal functioning of the particular organ.
In the methods of the invention, the manner in which the reporters are detected will depend upon the nature of the reporters.
Where the reporter is an entity detectable directly or indirectly in an imaging modality, then that modality may be used with images being generated of the whole animal or a region of interest therein or of a body fluid obtained therefrom. Whole body imaging is particularly desirable as it enables identification of library members which distribute to body sites not previously identified as potential targets and also enables identification of the specificity of the library members"" distribution. Thus where, for example, the target site of interest is the heart and two library members accumulate there, the preferred candidate may be the one with lesser accumulation in other tissues. Whole body imaging is feasible with many imaging techniques such as mr, X-ray (e.g. CT) and nuclear imaging.
Where the reporter is difficult to detect at a distance, or where the location is difficult to determine when there are significant amounts of tissue between reporter and detector, as is for example the case with light imaging techniques, it may be preferred to image only a region of interest within the body and even to insert the detector into the body through natural or surgically created apertures so as to ensure adequate proximity of detector and region of interest. Alternatively it may be desirable surgically to expose tissues or organs of interest, e.g. by removing skin, by opening the abdominal cavity or by removing part of the cranium.
Where the library members are to be detected in a body fluid, samples of this may be removed from the body and analysed, or blood may be continuously taken from the body, analysed and returned. In general, analysis of body fluids may be simpler than whole body or region of interest imaging as the fluids may be treated before analysis, e.g. to concentrate the reporter, to amplify the reporter, to remove non-reporter components (such as erythrocytes), etc. As a result, reporters which would not be detectable in vivo (e.g. since their identification requires amplification and sequencing or since their concentrations in vivo are too low or since their detection requires modification or destruction of their chemical environment or removal of surrounding tissue) may be used. Particularly preferably, the reporters used, where detection is by analysis of a body fluid, will be amplifiable reporters such as oligonucleotide xe2x80x9ctagsxe2x80x9d.
The method of the invention may be used to identify a drug candidate (optionally incorporating a reporter moiety) or a component, e.g. a targeting vector, for a drug candidate (again optionally incorporating a reporter moiety). Where a component is identified, the drug candidate will comprise this component bound to or otherwise associated with at least one further component, such as for example a therapeutically or diagnostically effective moiety which may be targeted to a desired body site by the identified component. Where the drug candidate is to comprise a reporter moiety, this may or may not be the same as the reporter moiety contained in the corresponding library member and indeed generally will be a reporter moiety detectable in vivo using a diagnostic imaging modality, e.g. in an X-ray (e.g. CT), mri, ultrasound, nuclear imaging (e.g. scintigraphy, PET or SPECT), magnetotomographic or light imaging modality.
In the methods of the invention, the combinatorial library is administered as a mixture of the library members to the living animal or living organ. This will generally involve administration of a solid, powder, syrup, solution, dispersion, suspension, spray, gel, emulsion, capsule or any other convenient dosage form by any convenient dosage route, e.g. parenterally or enterally for example into the nose, lungs, gastrointestinal tract, vagina, bladder, uterus, vasculature or muscles, or topical or subcutaneous administration. In general, administration will preferably be topical, subcutaneous, oral, rectal intramuscular, intra-arterial or intravenous.
Where xe2x80x9csuccessxe2x80x9d for a library member corresponds to a cross-barrier uptake, e.g. transdermal uptake, uptake from the gut, crossing of the blood brain barrier, etc., the administration route will preferably be direct to one side of the barrier in question, e.g. onto the skin or an exposed mucous membrane, into the gastrointestinal tract or into the vasculature, and detection of xe2x80x9csuccessxe2x80x9d will involve detection of library members which have crossed the relevant barrier, e.g. by passing into the blood following topical application or administration into the GI tract or by passing into the CSF following administration into the vasculature. Where xe2x80x9csuccessxe2x80x9d corresponds to accumulation in a particular organ or tissue, administration will generally be into the vasculature upstream of that organ or tissue, into the GI tract, to the skin, into muscle, into the lungs or directly into that organ or tissue. Where xe2x80x9csuccessxe2x80x9d corresponds to prolonged retention in or minimal uptake from a body duct or cavity (e.g. the vasculature), administration will generally be into that duct or cavity.
It will be apparent therefore that xe2x80x9csuccessxe2x80x9d depends upon the desired distribution of or affect of the library member. The contrast agent candidate to the selection of which the invention is directed is a diagnostic agent but in addition it may be a therapeutic or prophylactic drug, e.g. with one or more of the relevant properties being provided by a moiety which will be associated with a candidate component identified by the method of the invention.
Evaluation of xe2x80x9csuccessxe2x80x9d in sub-libraries where a previous library has achieved a desired effect may also include monitoring of physiological response (e.g. suppression of infection, anti-tumor activity, blood or other body fluid component analysis, blood pressure, cardiac function, etc) so that library members achieving a desired combination of effects or avoiding undesired effects may be identified.
xe2x80x9cSuccessxe2x80x9d may for example represent a desired targeting effect, a desired contrast effect, a desired therapeutic effect, etc. Thus a library member may for example comprise a first reporter moiety effective as a contrast agent in a particular diagnostic imaging modality, a tag unique to the member or a set of members and, optionally, a targeting moiety. In this instance xe2x80x9csuccessxe2x80x9d may be indicated by contrast effectiveness in the selected imaging modality with the tag being used as the means of establishing which reporter moiety or reporter/targeting vector combination enjoys this success.
Thus, in one embodiment, the method of the invention may be used to identify a vector moiety which may or may not be essentially physiologically inert in itself but which may be used to target a known effective therapeutic, prophylactic or diagnostic moiety to a body site of interest.
In an alternative embodiment, the methods of the invention may be used to identify contrast agent candidates or components thereof which are capable of detection (ie. may suitably be used as contrast agents in medical diagnostic imaging procedures) and which may have a desired physiological effect, and have the ability to reach a body site where they may exert that physiological effect or be detected. The invention is thus particularly suited for the identification of contrast agents which have a desired effect and are capable of reaching a desired body site from their administration site, e.g. tumor targeting diagnostic agents or combined therapeutic and diagnostic agents.
When one or more contrast agent drug candidates are selected using the methods of the invention, these contrast agent drug candidates, or a component or derivative thereof may be manufactured and if desired formulated with at least one pharmaceutically acceptable carrier or excipient. Such components of contrast agent drug candidates may for example be a targeting vector for the drug candidate. When such a component is identified the drug candidate may comprise this component bound to or otherwise associated with at least one further component, such as for example a therapeutically or diagnostically effective moiety. The term xe2x80x9cderivativexe2x80x9d as used herein includes a contrast agent drug candidate as identified in the methods of the invention but modified to incorporate a different reporter moiety or targeting vector. The term xe2x80x9cderivativexe2x80x9d also includes a modified contrast agent drug candidate wherein the reporter moiety is replaced by a therapeutic moiety.
Thus viewed from a further aspect the invention provides a process for the production of a contrast agent drug substance which comprises (i) selecting a contrast agent drug candidate or a component thereof using a selection method comprising a method according to the invention, (ii) optionally selecting a diagnostically or therapeutically effective drug component, (iii) manufacturing said contrast agent drug candidate or a contrast agent drug substance comprising the components selected in steps (i) and (ii), and (iv) optionally formulating the contrast agent drug candidate or contrast agent drug substance manufactured in step (iii) into a pharmaceutical composition together with at least one pharmaceutically tolerable carrier or excipient.
The library members may take a variety of forms thus in particular they may be molecules or molecular aggregates, e.g. vesicles or coated particles and they may or may not contain the oligomeric or decorated framework monomer structures typical of combinatorial chemistry libraries. Particularly preferably, the non-reporter part of the library members will be an oligomeric or decorated framework monomer structure typical of combinatorial chemistry libraries. Especially suitably, the non-reporter part is an oligopeptide, a peptoid, an oligosaccharide, an oligonucleotide or a non-polymeric small organic molecule (e.g. of molecular weight below 1000 D). The preparation of libraries of such materials has been widely described in the literature in the last decade (see for example M. Gallop et al. in J. Med. Chem., 37: 1233-1251 (1994); E. M. Gordon et al. in J. Med.Chem., 37: 1381-1401 (1994); M. C. Pirrung in Chemtractsxe2x80x94Organic Chemistry, 8: 5-12 (1995); W. J. Dower et al in Ann Rep in Med.Chem. 26: 271-280 (1991); W. H. Moos et al in Current Opinions in Structural Biology, 3: 580-584 (1993); M. R. Pavia et al. in Bioreg.Med.Chem. Lett., 3: 381-475 (1995); N. K. Terrett et al. in Tetrahedron, 51: 8135-8173 (1995); S. E. Blondelle et al. in Antimicrobial Agents and Chemotherapy, 40: 1067-1071 (1996); M. C. Desar et al. in Drug Development Research, 33: 174-188 (1994); D. J. Ecker in Biotechnology, 351-360 (1995); D. J. Gravert et al. in TIBTECH, 14: 110-112 (1996); D. Madden et al. in Perspectives in Drug Discovery Today, 1: 248-254 (1996); D. P. Curran in Chemtractsxe2x80x94Organic Chemistry, 9: 75-87 (1996); J. Eicher et al. in Molecular Medicine Today, 174-180 (1996); P. J. Schatz in Current Opinion in Biotechnology, 5: 487-494 (1994); J. Eicher et al. in Medicinal Research Reviews, 15: 481-496 (1995); S. Bormen in Chem. and Eng. News, February 12: 2954 (1996); M. Rinnova et al. in Collect. Czech.Chem. Commun, 61: 171-231 (1996); E. M. Gordon in Acc.Chem. Res., 29: 144-154 (1996); R. Cortese (Ed) in xe2x80x9cCombinatorial Libraries, Synthesis, Screening and Application Potentialxe2x80x9d (Water de Gruyter, 1996); A. M. Thayer in Chem. and Eng. News, February 12: 57-73 (1996); A. Furka et al. in Drug Development Research, 36: 1-12 (1995); M. H. Lyttle in Drug Development Research, 35: 230-236 (1995); A. W. Zarnik in Chemtractsxe2x80x94Organic Chemistry, 8: 13-18 (1995); and X-Willard in Eur. J.Med. Chem., 31: 87-98 (1996).
Such non-reporter components may readily be conjugated to a reporter or tag, e.g. by covalent bonding of a functional group at a terminus or on a side chain, or by conjugation to a moiety which will act as an anchor to the reporter, e.g. a lipophilic group which may associate with the membrane of a vesicle or an ionic group which can bond to the surface of an inorganic particle such as a magnetic metal oxide.
The reporter component may for example comprise a non-metal radioisotope, a metallated chelate complex or an oligopeptide or oligonucleotide covalently bonded to the non-reporter component, an organic chromophore or a particulate (e.g. a vesicle containing gas, a solid contrast agent or a contrast agent solution) to the surface of which is covalently or non-covalently attached the non-reporter component.